Update on Cassava Brown Streak Disease (CBSD) Status, prospects and implications for cassava projects
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Update on Cassava Brown Streak Disease (CBSD) Status, prospects and implications for cassava projects

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The Viruses of Cassava in Africa,CBSV epidemiolgy/transmission studies,Disease Management: Host resistance,Biotechnology Applications to Combat CBSD

The Viruses of Cassava in Africa,CBSV epidemiolgy/transmission studies,Disease Management: Host resistance,Biotechnology Applications to Combat CBSD

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Update on Cassava Brown Streak Disease (CBSD) Status, prospects and implications for cassava projects Update on Cassava Brown Streak Disease (CBSD) Status, prospects and implications for cassava projects Presentation Transcript

  • Update on Cassava Brown Streak Disease (CBSD)Status, prospects and implications for cassava projects J Legg E Kanju P Ntawuruhunga DJ Kim M Ferguson Lava Kumar MN Maruthi (NRI-UK) R4D Week, R&T MTP, 24 November09 www.iita.org
  • The Viruses of Cassava in AfricaGeminiviridae: Begomovirus•African cassava mosaic virus (ACMV)•East African cassava mosaic virus (EACMV)•South African cassava mosaic virus (SACMV)•EACMV-Cameroon, EACMV-Malawi, EACMV-Kenya, EACMV-ZanzibarIndian cassava mosaic virus*EACMV-Uganda (Recombinant virus) www.iita.org
  • The Viruses of Cassava in AfricaPoorly studied virusesCassava Ivorian bacilliform virus (Badnavirus)Cassava Kumi virusCassava ‘Q’ virusCassava common mosaic virus* (Potexvirus)Emerging virusCassava brown streak virusPotyviridae: Ipomovirus www.iita.org
  • Cassava brown streak disease www.iita.org
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  • Cassava brown streak diseaseCMD CBSD CMD+CBSD www.iita.org
  • CBSD: Pre-2000•Considered as ‘locally’ important problem1936: First report in northern Tanzania (Storey).1950: CBSD reported to be endemic on East African coast and Malawi.1995: Based on ‘pin-wheel’ inclusions potyvirus suspected (Harrison et al.).1995: Resistance/tolerance to CBSD identified in ‘local’ cassava cultivars in Tanzania. e.g. Nachinyaya.1998: High CBSD incidences (70%) along the northern Mozambique coast reported (Hillocks et al.) www.iita.org
  • CBSD: In new millennium • High incidence of CBSD in highlands • Increase in research efforts2000: Causal agent of CBSD identified as a potyvirus of genera Ipomovirus by a research group at Bristol University, UK (Monger et al.)2001-02: CBSV coat protein gene sequenced and RT-PCR-based diagnostic tool established for CBSV detection2003: Successful transmission of CBSD achieved with whitefly, Bemisia tabaci (Maruthi et al.)2004: Emergence of CBSD in Uganda (Alicai et al.) and Lake Zone area of Tanzania (Legg et al.), the first major outbreak at high altitudes www.iita.org
  • CBSD in epidemic era!2007 on wards: major initiatives to combat CBSD2008-Extensive disease incidence surveys initiated by (IITA)-Progress on resistance breeding and selections (IITA)-Development of improved RT-PCR diagnostics (many labs)-Development of monoclonal antibodies and ELISA (Winter et al.)2009• CBSV full genome sequence published (Mbazibwa et al.)• Occurrence of at least two strains of CBSV reported (Mbazibwa et al.)• Development of virus-resistant transgenic lines in N. benthamiana (Danforth)• Report of Spiraling whitefly as CBSV vector in Kenya (unpublished) www.iita.org
  • Towards sustainable CBSD Control Breeding / Agronomy / Extension (Conventional / MAS / transgenics) Plant Breeding Transgenic CBSD resistance Conventional & MAS Viral genes Resistant (RNAi) Varieties CBSV isolationBiochemical, Germplasm screening CBSD molecular & CBSV characterization Landraces and Control Biological hybrids Properties Diagnostic tools Disease Epidemiology Monitoring Virus isolates Quarantine Bioassays Vector biology Awarness Serological & Virus survival and spread Environmental factors Training Nucleic-acid assaysVirology / Pathology / Extension / NPPOs www.iita.org
  • CBSV: R4D statusConcept R4D stage at IITA• Characterization Advance• Genome sequencing Advance• Diagnostics Advance• Transmission Advance• Epidemiology (host-virus interaction) Advance• Conventional Resistance Advance Ham-1• Transgenics Developmental stage• Marker-assisted breeding Inception• Phytosanitation / awareness / training etc Adequate www.iita.org
  • CBSV: unique virus Ham-1 Source: S Winter, 2008 www.iita.org
  • CBSV inter-relationshipsUg KeTz 100 FN433933 CBSV Ma 43 2007 (Malawi:Salima) 79 FN433932 CBSV-Ma 42 2007 (Malawi:Chit... UG, Ken, Mal 92-95% 61 FN434109 CBSV-Ug 23 (full sequence) 2... 86-87% 70 FN433930 CBSV Kenya 125 1999 (Kenya:K... M 100 70-71% FN433931 CBSV-Ke 54 1997 (Kenya:Kilifi) 100 FJ185044. CBSV-Uganda (2006) NC 012698 CBSV isolate MLB3 full geno... FN434437 CBSV-Tan 70 (full sequence) ... 79-80% Tz, Moz FN434436 CBSV-Mo 83 (full sequence) 2... 96% 100 100 GQ329864 CBSV-Tz (full sequence) 200... NC 006941 CVYV0.1 NJ Tree of full-length CBSV genomes www.iita.org
  • CBSV inter-relationships 62 FN434109 Uganda Ug-23 97 EU916829 Uganda (LWR2) 100 EU916828 Uganda (HMA9) EU916830 Uganda (IGA8)Ug Ke 87 EU916827 Uganda_Nam (NTG10) 87 57 EU916832 Uganda-Busia (BSA4) 91-100% EU916831 Uganda-Busia (BSA2)Tz 71 FJ185044 Uganda-2006 FN433932 Malawi 2007 (Chitipa) 99 99 FN433933 Malawi 2007 (Salima) FN433930 Kenya 125 1999 69-71% EU916826 Tanzania (MLB9) 100 NC 012698 Tanzania MLB3 M 100 EU916825 Tanzania (MLB3) FN433931 Kenya 54 1997 (Kilifi) 65 FN434436 Moza Mo-83 GQ329864 Tanzania FN434437 Tanzania- Tan70 AY008440 Tanzania (type C) 91-100% 99 AY007597 Tanzania TZC2 98 AY008441 Tanzania (type B) 94 FJ821794 Tanzania (KBH2) FJ821795 Tanzania (KBH1) 95 AY008442 Tanzania (type A) AF311053 Mozambique MZQ1 75 97 AF311052 Mozambique MZQ2 NC 006941 CVYV NJ Tree of full-length CBSV coat protein gene www.iita.org
  • Symptom diversitySource: MN Maruthi, NRI-UK www.iita.org
  • CBSD DiagnosticsCBSV and CMBVs are complex, often necessitating multiple tests. Cassava brown streak virus •Sequence information points to divergent types (species complex!) Cassava mosaic begomoviruses (CMBVs) in SSA • African cassava mosaic • East African cassava mosaic • East African cassava mosaic Cameroon virus • East African cassava mosaic Zanzibar virus • East African cassava mosaic Malawi virus • East African cassava mosaic Kenya virus • East African cassava mosaic virus-Uganda • South African cassava mosaic virus • Indian cassava mosaic www.iita.org
  • Multiplex PCR for CBSV & CMBV CBSV-S1/S2 + CMB CBSV-L1/L2 + CMB Sap DNA & RNA Sap DNA&RNA M1 2 3 4 5 6 7 1 2 34 5 6 7 M1 2 3 4 5 6 7 1 2 34 5 6 7 MEACMV Lanes 1 to 4: CBSV infected samplesACMV Lane 5: Healthy cassava Lane 6: CMD infected cassavaCBSV Lane M: Molecular weight marker (100 kb ladder) CBSV-D1/D2 + CMB Sap DNA & RNA • Primers CBSV-S1/S2 and CBSV-L1/L2 M1 2 3 4 5 6 7 1 2 3 4 5 6 7 M resulted in expected product size in EACMV multiplex assay with CMBV primers. ACMV • But, CBSV-D1/D2 did not result in amplification. www.iita.org
  • CBSV Sensitivity assay CBSV-L1/L2 + CMB CBSV-S1/S2 + CMB M 1 2 3 4 5 6 7 8 9 10 11 M 1 2 3 4 5 6 7 8 9 10 11 Lane1 = undiluted sap; Lane 2 = 1:10; Lane 3 = 1:100; Lane 4 = 1:1000 (v/v)Ten fold serial dilution of sap extract (1:20 w/v) up to 10-10 dilution (v/v).•CBSV detected consistently in sap diluted up to 10-3.•Sometimes detection obtained even at 10-6 dilution. www.iita.org
  • CBSV Composite Assay•Sap extracted from composite sample comprising of 9 healthy leaves and oneinfected leaf•Sap extract from 100 mg tissue (1:20 w/v) used as template for RT-PCR CBSV-L1/L2 + CMB Sample 1 Sample 2 M 1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 10 M•Sap extract (1:20 w/v) from composite sample in 10 fold serially diluted upto 10-10 dilution. CBSV detected in sap diluted up to 10-6. www.iita.org
  • • Adequate ‘basic’ knowledge and technologies are available that can contribute to studies on CBSD epidemiology and development of host resistance and more. www.iita.org
  • CBSD Epidemiology J Legg www.iita.org
  • CBSV epidemiolgy/ transmission studies• Components – Macro epidemiology. Through use of historical and new surveillance data from East/Central Africa – Micro CBSV epidemiology CBSV, B. tabaci population dynamics trials – Kibaha (Tz) – B. tabaci control trials – Ukerewe (Tz) and Namulonge (Ug) – B. tabaci transmission studies – Kibaha and NRI – Alternative host and alternative vector studies (J. Ndunguru, ARI Mikocheni, Tanzania) www.iita.org
  • CBSV Vector(s) ?Whitefly Spiraling whitefly(Bemisia tabaci) (Aleurodicus dispersus) www.iita.org
  • ‘Micro’ CBSV Epidemiology/ B. tabaci population dynamics• Planting Material. Visually CBSD-free cv. Kiroba from relatively unaffected Mkuranga District• Virus Testing. Leaves from all parent stems tested for CBSV using RT-PCR. Only negatives used• Planting. November. In four separate locations at Kibaha isolated from other cassava fields• Data Collected. Weekly records of CBSD incidence, severity and whitefly adult abundance. Samples collected www.iita.org
  • CBSV EpidemiologyExperimental Layout 5 4 3 2 1 Spreader Initially CBSD-free plots Prevailing Wind www.iita.org
  • CBSD incidence (%) 12 / 29 /2 0 10 20 30 40 50 60 70 80 90 100 1/ 008 12 /2 1/ 009 26 /2 0 2/ 09 9/ 2 2/ 009 23 /2 0 3/ 09 9/ 2 3/ 009 23 /2 0 4/ 09 6/ 2 4/ 009 20 /2 0 5/ 09 4/ 2 5/ 009 18 /2 0 6/ 09 1/ 2 6/ 009 15 /2 6/ 009 29 Incidence /2 7/ 009 13 New infection /2 7/ 009 27 /2 8/ 009 10 /2 00 9 0 5 10 15 20 25 30 CBSD Disease Progress - Kibahawww.iita.org
  • CBSD, Whiteflies - Kibaha www.iita.org
  • CBSD/whitefly relationship 16 2.5 Whitefly abundance 3 wks previous 14 CBSD transformed infection CBSD transformed infection 2 12Whitefly abundance y = 0.048x – 0.018 10 (F = 13.3; r2 = 0.63; P < 0.001) 1.5 8 1 6 4 0.5 2 0 0 1 2 3 4 5 6 7 8 Week www.iita.org
  • Whitefly Control and CBSV Epidemiology• Experimental Locations. High CBSD pressure locations in Uganda (Namulonge) and Tanzania (Ukerewe Island)• Planting Material. Visually CBSD-free cvs. TMS I92/0057 and Njule (Uganda) and TMS I92/0057 and Liongo Kwimba (Tanzania) collected from CBSD-unaffected locations• Planting. December 2008. Replicated trial at each site• Treatments. Whitefly controlled in ‘whitefly-free’ plots through combined use of Imidacloprid (soil drench) and Cypermethrin (foliar application)• Virus Testing. Samples collected from 10 plants per plot for each site in May (Tanzania) and June (Uganda)• Data Collected. Monthly records of CBSD incidence, severity and whitefly adult abundance www.iita.org
  • Whitefly Control and CBSV Epidemiology Whitefly Variety Plants CBSD CBSV W’fly CMD treatment (%) (%)Uganda Treated I92/0057 163 53.4 - 3.4 6.8 Untreated I92/0057 165 95.8 - 127.6 25.5 Treated Njule 165 50.9 - 2.0 13.3 Untreated Njule 167 97.6 - 113.4 100Tanzania Treated I92/0057 60 86.7 22.5 0.6 0 Untreated I92/0057 62 90.3 45 94.9 0 Treated Liongo 58 69 17.5 0.6 58.6 Untreated Liongo 48 100 67.5 87.2 97.9 www.iita.org
  • Whitefly-treated Njule Whitefly-infested NjulePhoto: G. Okao-Okuja www.iita.org
  • Cassava Virus Spread, Whitefly Abundance cv. Liongo Kwimba - Tanzania 0.6 180 CBSD CMD 160 0.5 Whiteflies 140Newly Diseased Proportion 0.4 120 Whitefly abundance 100 0.3 80 0.2 60 40 0.1 20 0 0 1 2 3 4 5 6 7 8 Months after Planting
  • Cassava Virus Spread, Whitefly Abundance cv. Njule - Uganda
  • Whitefly Control and CBSV Epidemiology - Observations• Planting material. Can’t be 100% sure of CBSV-free status. Have established isolated CBSD-free multiplication sites in the Usambara Mountains, eastern Tanzania• Diagnostics. Cheaper and more reliable methods essential for further work• Results. Suggest an association between CBSD and whitefly populations, contrasting with CMD• Transmission. Results suggest whitefly transmission through a non-persistent mechanism• Climate interaction. Appears that disease will only be expressed when climatic conditions are favourable – drought and high temperature www.iita.org
  • Digital Early Warning Network (DEWN) Piloting in TanzaniaAugust 2009 September 2009 www.iita.org
  • Smart Surveillance SystemsDigital Forms Digital Images Digital Maps www.iita.org
  • Disease Management: Host resistanceEdward Kanju and Pheneas Ntawuruhunga www.iita.org
  • Disease Management: Host resistance• Training: technicians on cassava grafting to test highly promising clones for resistance to CBSD• Grafting trials: established using clones that have remained disease symptom free• Multiplication: promising breeding lines• Technical backstopping: of NARS on field screening of cassava clones for resistance/tolerance to major diseases and pests (through PVS)• Regional exchange: improved germplasm (with KEPHIS) www.iita.org
  • Cassava improvement goal :To develop varieties which combine high and stable yield with good quality characteristics for end users Dry matter yield/Unit/Time; Disease/pest resistance Traits Nutritional and end users quality Stress (drought) tolerance Vectors Enabling Technologies Promoters Selectable markersGermplasm that respond to end-users’ needs The most effective and realistic approach to reducing losses to CMD and CBSD is the use of host-plant resistance or deployment of less- susceptible cultivars www.iita.org
  • Grafting Trial Tanzania• Seven participants were trained in germplasm screening.• The following clones/breeding lines were grafted at ARI Chambezi (Bagamoyo district) and ARI Naliendele (Mtwara district): – KBH 06/98 – KBH 02/066 – KBH 02/363 – KBH 01/110 – KBH 06/18 – KBH 06/12• > 80% successful grafts• The clones are being multiplied at ARI Chambezi (4 are tested on- farm) www.iita.org
  • Promising clones• Performance of 963 cassava genotypes under high CBSD pressure at Namulonge (Uganda) for 3 years. CBSD Disease Group No. genotypes Percentage index [Set 1 and 2] (%)0 Resistant 410 42.60 – 20 Moderately resistant 172 17.9>20 – 57.5 Moderately susceptible 186 19.3>57.5 – 150 Susceptible 118 12.3>150 Highly susceptible 77 8.0• From further screening in 2008, 15 best genotypes selected for dissemination through GLCI project. www.iita.org
  • Promising clonesRef.# Clone Quantity Ref.# Clone Quantity 1 MM06/0135 8 8 MM06/0131 8 2 MM06/0124 4 9 MM06/0012 5 3 MM06/0076 2 10 MM06/0019 6 4 MM06/0139 5 11 MM06/045 3 5 MM06/0013 3 12 MM06/0112 4 6 MM06/0011 3 13 MM06/0023b 4 7 MM06/0024 5 14 MM06/0079 9 www.iita.org
  • CMD and CBSD resistant cassava• A second screening trial conducted from botanical seeds developed at IITA-Tanzania (Amani sources).• From this, 8 best clones have been identified from Mukono trials which were identified and selected also by farmers.• In total 52 genotypes have been selected as best potential resistant clones to both CBSD and CMD. These have been transferred to KEPHIS for cleaning, and tissue culture for exchange with NARS. www.iita.org
  • Crosses produce seeds Cassava BreedingTransplantation Scheme Seedlings are evaluated for their reaction to diseases and pests. Clones selected are advanced for screening for further activities in different agro-ecologies Seedlings www.iita.org
  • CBSD tolerant clones for Burundi360 seedlings fromTanzanian CBSDtolerant parents www.iita.org
  • New Varieties released in ZanzibarIt takes 7 – 8 years to develop a new variety www.iita.org
  • Dual resistant clones in Uganda? Clone Pedigree1. MM Kitumbua-OP 06/00132. MM Kigoma Red- 06/0046 OP3. MM Kigoma Red- 06/0074 OP4. MM Kibaha-OP 06/00825. MM Kibaha-OP 06/00836. MM Kibaha-OP Eight clones have remained CMD and CBSD symptom-free under 06/0138 high pressure for three seasons. They will be challenged by grafting7. MM Kibaha-OP www.iita.org
  • ‘Biotechnology Applications to Combat CBSD’Agreement signed: Nov 5th 2009Implementing agency: IITAPartner institutes: NARO, ARI –TanzaniaCollaborating institutes: DDPSC, BecA, ILRIBudget: Approx $2.4m over four yearsAssociated project: Cassava Genomics: bridging the gap betweensequence and breeding applications www.iita.org
  • Research Objectives1. Improve existing markers for one source of CBSD resistance/tolerance (SNP development and fine mapping in Namikonga)2. Develop new markers and breeding resources for six new sources of resistance/tolerance to CBSD3. Utilize markers in Tanzania and Uganda to begin breeding new CBSD-resistant cultivars suitable for the region (exit strategy)4. Test a transgenic approach to controlling CBSD and move the most promising transgene into a popular Ugandan cultivar (CFT and transformation of Ugandan varieties) www.iita.org
  • Training & Capacity•Development of surveillance protocols•Training in field surveillance www.iita.org
  • Regional Training for the Disease Objective of GLCICassava Viruses: Biology, Diagnostics and Management 28 October – 6 November 2009, IITA, Dar es Salaam, Tanzania www.iita.org
  • Future priorities• Consolidation and coordination of existing efforts• Upstream research to address recalcitrant issues (eg. Combine resistance to CMD, CBSD and whitefly)• Framework for improved cassava germplasm distribution to partners (MTA / sMTA / IP Issues) www.iita.org
  • Thank you www.iita.org